Design, synthesis and biological evaluation of: N -arylsulfonyl carbazoles as novel anticancer agents

Article abstract of DOI:10.1039/c8ra02939c

In this work, a set of structurally diverse synthetic carbazoles was screened for their anticancer activities. According to structure-activity relationship studies, carbazoles with an N-substituted sulfonyl group exhibited better anticancer activity. Moreover, compound 8h was discovered to show the most potent anticancer effects on Capan-2 cells by inducing apoptosis and cell cycle arrest in G2/M phase. Finally, the in vivo study demonstrated that 8h prevented the tumor growth in PANC-1 and Capan-2 xenograft models without apparent toxicity.

Full text of DOI:10.1039/c8ra02939c

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PAPER
Design, synthesis and biological evaluation of
N-arylsulfonyl carbazoles as novel anticancer
agents†
Cite this: RSC Adv., 2018, 8, 17183
Xin You,‡a Daqian Zhu,‡ab Wenhua Lu,a Yichen Sun,a Shuang Qiao,ab Bingling Luo,ab
b
a
a
ab
Yongliang Du,ab Rongbiao Pi, Yumin Hu, Peng Huang and Shijun Wen
*
*
In this work, a set of structurally diverse synthetic carbazoles was screened for their anticancer activities.
According to structure–activity relationship studies, carbazoles with an N-substituted sulfonyl group
exhibited better anticancer activity. Moreover, compound 8h was discovered to show the most potent
anticancer effects on Capan-2 cells by inducing apoptosis and cell cycle arrest in G2/M phase. Finally,
the in vivo study demonstrated that 8h prevented the tumor growth in PANC-1 and Capan-2 xenograft
models without apparent toxicity.
Received 5th April 2018
Accepted 30th April 2018
DOI: 10.1039/c8ra02939c
rsc.li/rsc-advances
synthetic synthons for complex polycyclic arenes.11–20 Due to the
structural similarity of carbazoles and CDIs, it was envisioned
1. Introduction
that the insertion of nitrogen into the CDIs would be able to
afford carbazoles. Indeed, we previously developed a synthetic
strategy for carbazoles via the reaction of CDIs with various
amines which was mediated by Cu(OAc)2 at low cost.11 With this
strategy, an in-house chemical library of carbazoles with struc-
tural diversity and complexity was successfully built. The
previous biological neuroprotective screening of the carbazole
library found that some N-phenyl carbazoles showed good neu-
roprotection of neuronal cells HT22 against cell injury induced by
glutamate or homocysteic acid.12 Pancreatic cancer is one leading
cause of cancer-related death and remains a serious clinical
problem due to poor prognosis and the limited drug options.21 In
the continuation of our efforts to discover novel and potent
anticancer agents, our in-house synthetic carbazoles were further
screened for their potential antiproliferation of pancreatic cancer
cells. The screening assay provided a structural starting point for
further structure–activity study. In this work, we report the design
and synthesis of structurally diversied carbazoles and their
biological evaluation, leading to the discovery of novel N-aryl-
sulfonyl carbazoles with both in vitro and in vivo antitumor
therapeutic activities.
Carbazoles and their derivatives are of great interest in the
research elds of synthetic chemistry and medicinal chemistry,
due to their tricyclic structure and widespread pharmacological
properties.1 In general, carbazoles constitute a useful class of
polycyclic aromatic drugs displaying diverse activities including
antibacterial,2 neuroprotective,3 anti-inammatory,4 antioxi-
dant,5 and anti-HIV functions.6 More importantly, carbazoles
are found to have effective antiproliferative activity on tumor
cells (Fig. 1). Clausine B, a naturally occurring carbazole alka-
loid, was found to be active (IC50 < 30 mg mLꢀ1) against four
human cancer cell lines including MDA-MB-231 (breast cancer),
HeLa (cervical cancer), CAOV3 (ovarian cancer) and HepG2
(hepatic cancer).7 Clausenawallines
F showed cytotoxicity
against oral cavity cancer (KB) and small-cell lung cancer (NCI-
H187) with IC50 values of 10.2 mM and 4.5 mM, respectively.8 Due
to the important pharmacological activities of the reported
natural occurring carbazoles, an array of compounds contain-
ing carbazole motifs has been designed and synthesized for
biological evaluation. For example, LY-333531 was reported as
an anticancer agent by inhibiting Pim-1 kinase with IC50 value
of 0.2 mM.9,10
In the recent years, other research groups and ours have
demonstrated that cyclic diphenyliodoniums (CDIs) are useful
aSun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South
China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen
University, 651 Dongfeng East Road, Guangzhou 510060, China. E-mail: wenshj@
sysucc.org.cn; huangpeng@sysucc.org.cn
bSchool of Pharmaceutical Sciences, Sun Yat-sen University, 132 Waihuan East Road,
Guangzhou 510006, China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c8ra02939c
Fig. 1 Representative carbazoles with anticancer activities.
RSC Adv., 2018, 8, 17183–17190 | 17183
‡ Both authors contributed equally to this work.
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Scheme 1 Synthesis of carbazoles. Reaction conditions: diphenyleniodonium 5 (0.1 mmol), amine 6 (4 equiv.), Na2CO3 (3 equiv.), Cu(OAc)2 (0.2
equiv.), 16 h, in refluxing i-PrOH/(CH2OH)2 (1.8/0.2 mL), argon atmosphere. (a) Additional CuI (0.2 equiv.) was added. (b) 7o was obtained from the
desulfonylation of 8a.
2. Results and discussion
2.1 Chemistry
Table 1 The primary antiproliferative screening of 7a–7o and 8a–8f
on PANC-1 and Capan-2 pancreatic cancer cell lines
2.1.1 General procedures for the synthesis of carbazoles
with cyclic diphenyliodoniums. In previous work, we have
demonstrated the construction of N-substituted carbazoles
starting from cyclic diphenyl iodoniums.11 The procedures for
the synthesis of carbazoles were depicted in Scheme 1. The
reactions of cyclic diphenyl iodoniums 5 with amines 6 were
performed in a reuxing isopropanol/ethylene glycol (9/1)
mixed solvent at argon atmosphere for 16 h, with Cu(OAc)2 as
the catalyst and Na2CO3 as the base. Thus, the reactions effec-
tively afforded a series of N-aryl (7a–7i and 7l–7m), N-pyridinyl
(7n), N-aliphatic (7j, 7k) and N-arylsulfonyl (8a–8f) substituted
carbazoles at moderate to good yields. Besides, carbazole (7o)
was also accessible aer desulfonylation of 8a. A mechanism
study for this conversion was reported in our previous work.11,12
Inhibitory rate of
PANC-1 viability (%)
Inhibitory rate of
Capan-2 viability (%)
Compound
30 mM
10 mM
30 mM
10 mM
7a
7b
7c
7d
7e
7f
7g
7h
7i
25.02
12.59
22.8
25.9
43.5
20.38
16.42
18.61
17.65
24.4
31.48
21.01
20.65
34.8
25.25
23.82
15.85
12.91
20.56
28.82
11.75
18.58
20.05
32.61
33.72
30.36
38.41
20.91
27.03
61.57
66.79
57.72
72.04
28.39
66.6
49.29
22.55
20.32
38.12
14.53
15.93
35.65
35.98
42.79
24.94
ND
67.81
70.54
69.39
72.62
28.46
82.2
11.75
3.8
34.27
3.81
71.1
19.05
25.61
18.18
36.68
23.76
13.16
33.84
36.27
41.55
63.98
54.71
34.94
61.2
54.31
19.78
44.34
5.4
9.61
34.73
35.9
43.66
59.8
54.17
53.92
58.6
7j
7k
7l
7m
7n
7o
8a
8b
8c
8d
8e
8f
2.2 Biology
2.2.1 In vitro anticancer screening. The in vitro antitumor
activities of the synthesized carbazole derivatives against
human cancer cell lines, including two pancreatic cancer cell
lines (PANC-1 and Capan-2), were assayed by MTS method. A
summary of these results was shown in Table 1. N-Phenyl
substituted carbazoles were found to display moderate to good
neuroprotective activity by mediating ROS decrease in the
neuronal cells HT22.12 However, in this context, it was observed
that the treatment with most of N-phenyl (7a–7i and 7l, 7m), N-
56.67
48.05
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gaining antiproliferative activities. Carbazole with
a tri-
uoromethyl (8f) was the best compared to the ones with
alternative functional groups such as 8a, 8c (OMe), 8b and 8d
(phenyl) and 8e (CN).
2.2.2 Optimization of compound 8f. Based on the structure
of 8f (Scheme 2), a preliminary structure–activity relationship
(SAR) study was conducted in order to nd more potent agents,
as shown in Table 2. In terms of substituents on the arylsul-
fonyl, tert-butyl (t-butyl, 8g), methoxy (OMe, 8h), and cyano (CN,
8m) enhanced the anticancer efficiency. On the contrary, the
carbazoles with an N-arylsulfonyl substituted by uorine (F, 8i),
chlorine (Cl, 8j) and triuoromethyl (CF3, 8l) lost their anti-
cancer efficacy drastically. Interestingly, the carbazole with an
N-arylsulfonyl substituted by bromine (Br, 8k) showed more
effective cytotoxicity than its counterparts 8i (–F) and 8j (–Cl). A
thiophenyl group to replace phenyl of arylsulfonyls did not help
to improve the inhibitory effect of 8n compared to the leading
compound 8f. It is worth mentioning that 8g–8n were charac-
terized by NMR and HRMS. Finally, the most potent compound
8h was taken for further investigation for its antiproliferation
against four different types of human cancer cell lines PANC-1
Scheme 2 Newly synthetic arylsulfonyl N-substituted carbazoles for
more potent anticancer agents.
Table 2 The antiproliferative evaluation of 8g–8n on PANC-1 and (pancreatic cancer), Capan-2 (pancreatic cancer), SGC-7901
Capan-2 pancreatic cancer cell lines
(gastric cancer), HCT-116 (colon cancer), and HL-60
(leukemia), while 5-uorouracil (5-FU) was selected as a posi-
Inhibitory rate of
Inhibitory rate of
tive control. As shown in the Table 3, 8h demonstrated a broad
spectrum of antitumor activities. Under our experiment condi-
tions, the antiproliferative activities of 8h were found to be more
potent than 5-FU. Moreover, compound 8h showed much lower
toxicity (IC50, 20.04 mM) to normal HFL-1 broblast cell line,
indicating a good selectivity between human cancer and normal
cells.
2.2.3 The effect of 8h on cell cycle of Capan-2 cancer cells.
As a planar and aromatic polycyclic compound, carbazole
derivative 8h was reasoned to interact with DNA and induce
a DNA damage.23 Indeed, the treatment with 8h in PANC-1 and
Capan-2 cells increased the level of g-H2AX protein, which is
a sensitive molecular marker of DNA damage (Fig. 2A).
Considering that DNA damage has been reported to induce cell
cycle arrest,24–26 the effect of 8h on cell cycle distribution was
also studied at different concentrations on Capan-2 cells. As
shown in Fig. 2B–C, the treatment of Capan-2 cells at 5 mM
resulted in more than 90% of the cells arrested in G2/M phase.
The substantial accumulation of cancer cells in G2/M phase
induced by 8h might contribute to the antiproliferation of the
compound.
PANC-1 viability (%)
Capan-2 viability (%)
Compound
30 mM
10 mM
30 mM
10 mM
8g
8h
8i
8j
8k
8l
35.5
72.2
5.6
36.8
53.9
43.3
76.9
61.6
25.1
68.2
13.42
16.5
54.8
8.49
59.9
57.3
36.1
86.8
19.55
58.7
84.9
29.7
80.2
40.4
44.1
89.4
14.4
52.4
73.3
31.41
64.0
49.1
8m
8n
pyridinyl (7n), N-aliphatic substituted carbazoles (7j, 7k) and
carbazole 7o led to an inhibition of cell viability less than 50% at
the tested concentrations. Thus, compounds 7a–7o were
excluded in further study, which was based on criterion of NCI-
60 program.22 Meanwhile, most of the N-arylsulfonyl
substituted carbazoles (8a–8f) exhibited potent cytotoxic activi-
ties against PANC-1 and Capan-2 cell lines. Furthermore,
among these effective carbazoles, different substituents on the
carbazole motif and the N-sulfonyl group played a crucial role in
Table 3 In vitro IC50 value (mM) of 8h against tumor cell lines and normal cell line using MTS assaya
Cell line (IC50 in mM)
Compound
PANC-1
Capan-2
SGC-7901
HCT-116
HL-60
HFL-1
8h
5-Fu
3.69 ꢁ 0.30
1.30 ꢁ 0.28
2.25 ꢁ 0.21
1.90 ꢁ 0.14
2.20 ꢁ 0.70
20.04 ꢁ 2.48
19.31 ꢁ 2.76
32.40 ꢁ 3.70
16.56 ꢁ 1.32
10.03 ꢁ 0.88
NT
NT
a
NT: not tested. Values are expressed as mean ꢁ standard deviation (SD) of three independent duplicate experiments.
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Fig. 2 Compound 8h induced DNA damage and cell cycle arrest in
G2/M phase. (A) Protein g-H2AX was analyzed by western blot. PANC-
1 and Capan-2 cells were treated with 1 or 3 mM of compound 8h for
24 h. (B) Effect of compound 8h at 3 mM or 5 mM on the cell cycle of
Capan-2 for 24 h. Cellular DNA contents were measured by PI staining
using flow cytometry. The numbers indicate the normalization of each
phase of cell cycle. (C) Histograms demonstrated the percentage of
Capan-2 at different phases of the cell cycle. Error bars represent
mean ꢁ SD. *p < 0.05; **p < 0.01; ***p < 0.001.
Fig. 3 The effect of compound 8h on the level of cyclin B1, p-Tyr15-
cdc2. (A) Western blot was used to determine the expression of cyclin
B1, p-Tyr15-cdc2 in PANC-1 and Capan-2 cells treated in the absence
or presence (1 or 3 mM) of 8h for 24 h. (B) Densitometric analysis of
cyclin B1, p-Tyr15-cdc2 proteins normalized with b-actin. The
expression ratio of related proteins was quantified against b-actin
using ImageJ software. Data were derived from three independent
replicates. Bars means ꢁ SD. *p < 0.05; **p < 0.01; ***p < 0.001.
2.2.4 The effect of 8h on the levels of cell-cycle regulatory
proteins. To explore the potential molecular mechanism for 8h-
induced cell cycle arrest at the G2/M phase, we examined the
protein expression of cyclin B1 and phospho-Tyr15-Cdc2 which
are crucial regulators in G2/M phase transition.27,28 It is well
known that the cell cycle is primarily regulated by CDK-cyclin
complexes containing cyclin-dependent kinases (CDKs) and
their cofactor cyclins.29 The progression from G2 to mitosis is
driven by the formation and activation of cdc2 (CDK1)-cyclin B1
complex.30 As one of cdc2 negative regulator sites, Tyr15 phos-
phorylation inhibits cdc2 activity.27 In PANC-1 and Capan-2
cells, 8h signicantly decreased the expression level of cyclin
B1 and increased the expression level of phospho-Tyr15-cdc2
(Fig. 3A–B), thus favoring cell cycle arrest at G2/M phase.
2.2.5 The effect of 8h on the apoptosis induction of Capan-
2 cancer cells. We further investigated whether an induced
apoptosis contributed to 8h's lethality by Annexin V-FITC and PI
staining using ow cytometer. The apoptotic effect of
compound 8h (3 mM and 5 mM) was evaluated on Capan-2 cells.
Data indicated that 8h signicantly increased the percentage of
apoptotic cells in a dose-dependent manner compared to the
control (Fig. 4A–B).
Fig. 4 Flow cytometric analysis of 8h induced apoptosis in Capan-2
cells using Annexin V-FITC and PI double staining. (A) Apoptosis rates
were determined after Capan-2 cells were treated with 8h at 3 mM or 5
mM for 48 h. Percentage numbers indicate cell death population. (B)
Percentage of Annexin V/PI positive cells were derived from three
independent replicates. Data shown are mean values ꢁ SD (error bar).
**p < 0.01; ***p < 0.001.
2.2.6 The role of ROS in mediating 8h-induced apoptosis.
Reactive oxygen species (ROS) are well known to be a major
player in redox signaling and
a critical mediator of
apoptosis.31,32 The cancer cells in advanced stage tumors oen
exhibit high oxidative stress, suggesting that it might be
possible to preferentially kill these cancer cells by
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Fig. 5 Induction of ROS increase by compound 8h and its partial
reversion by an antioxidant NAC. (A) DCF-DA staining showed the
effect of 3 mM (blue line) and 5 mM (red line) of 8h on cellular ROS levels
after 24 h treatment. (B and C) Pretreatment with NAC effectively
reversed the ROS accumulation. Capan-2 cells were pre-treated with
3 mM NAC for 1 h and then incubated with compound 8h (3 or 5 mM)
for 24 h. Error bars represent mean ꢁ SD. (D and E) Compound 8h-
induced apoptosis in Capan-2 cells was partially prevented by pre-
treatment with NAC compared to Fig. 4. Capan-2 cells were pre-
treated with 3 mM NAC for 1 h, and then incubated with the indicated
concentrations (3 or 5 mM) of 8h for 24 h. Error bars represent mean ꢁ
SD. *p < 0.05; **p < 0.01; ***p < 0.001.
Fig. 7 In vivo antitumor effect of compound 8h in Capan-2 xenograft
model. 8h was administered three times per week intraperitoneally at
40 mg kgꢀ1 dosage. (A) Image of all dissected tumors. (B) The effect of
8 h on the Capan-2 tumor growth. (C) Tumor weight after 25 days of
treatment. (D) Mice weights were measured twice a week. Bars means
ꢁ SD. *p < 0.05.
marked protection against 8h-induced apoptosis (Fig. 5D–E),
suggesting that the ROS generation induced by 8h might play an
important role in the cell apoptosis.
2.2.7 In vivo anticancer activity of 8h. Based on the prom-
ising in vitro anticancer activity of 8h toward various cell lines,
especially pancreatic cancer cell lines, we next evaluated in vivo
anti-cancer activity of 8h in PANC-1 and Capan-2 xenogra
mice. Compared with the control group, PANC-1 tumor growth
was substantially reduced in the mice treated with 8h intra-
peritoneally three times weekly at a 40 mg kgꢀ1 dosage, with
approximately 40% volume inhibition (Fig. 6A–B). Furthermore,
the treatment with 8h for 28 days signicantly diminished
tumor weight (Fig. 6C). Meanwhile, no signicant weight loss or
illness was observed in the treatment group (Fig. 6D). Moreover,
the anticancer effect of 8h was also observed in another
pancreatic cancer cell line Capan-2 xenogra mice (Fig. 7).
Taken together, 8h exhibited signicant in vivo antitumor effi-
cacy at a safe dose.
Fig. 6 In vivo antitumor effect of compound 8h in PANC-1 xenograft
model. 8h was administered three times per week intraperitoneally at
40 mg kgꢀ1 dosage. (A) Image of all dissected tumors. (B) The effect of
8h on the PANC-1 tumor growth. (C) Tumor weight at day 28 after 4
weeks of treatment. (D) Mice weights were measured twice a week.
Bars means ꢁ SD. **p < 0.01; ***p < 0.001.
3. Conclusions
In conclusion, a chemical library of N-substituted carbazoles
was synthesized conveniently, and all the compounds were
screened for a potential anticancer activity. Results demon-
strated that the N-arylsulfonyl substituted carbazoles showed
signicant antiproliferative ability. A preliminary SAR study
found that the synthetic compound 8h displayed the most
potent cytotoxic activity against PANC-1, Capan-2, SGC-7901,
HCT-116, HL-60 cancer cells. Moreover, compound 8h exhibi-
ted promising in vivo anticancer therapeutic efficacy in PANC-1
and Capan-2 xenogra mice models without an apparent side
effect. The following preliminary investigation of its action
model mechanism implied that the ROS mediated-apoptosis as
well as the G2/M cell cycle arrest might contribute to the anti-
cancer activity of 8h. Environmentally benign and easily
pharmacologically increasing the ROS level transiently.33 In
previous work, we demonstrated that such strategy was effective
to eliminate cancer cells by mediating intracellular ROS
increase.34,35 Therefore, it was of our interest whether such N-
arylsulfonyl carbazole 8h could elevate the intracellular ROS
level in cancer cells. To test this possibility, the ROS level was
measured when Capan-2 cells were subjected to 8h. As shown in
Fig. 5A–C, 8h caused a concentration-dependent increase of
cellular ROS, which could be partially reversed by pretreatment
with an antioxidant N-acetyl-L-cysteine (NAC), a scavenger of
ROS. Importantly, pretreatment with NAC also resulted in
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The general procedure for the preparation of these carba-
zoles was exemplied by the synthesis of 8h:
To a stirred solution of 3-(triuoromethyl)dibenzo[b,d]iodol-
5-ium triuoromethanesulfonate (100 mg, 0.2 mmol, 1 eq.) in i-
PrOH (1.8 mL) and ethylene glycol (0.2 mL), was added 4-
methoxybenzenesulfonamide (151 mg, 0.8 mmol, 4 equiv.),
sodium carbonate (64 mg, 0.6 mmol, 3 equiv.), Cu(OAc)2 (8 mg,
0.04 mmol, 0.2 equiv.). The reaction proceeded at a reux for
16 h under argon atmosphere before i-PrOH was removed by
a rotary evaporation. The remained mixture was partitioned
between water and EtOAc, and the aqueous phase was extracted
with EtOAc. The combined organic phases were washed with
H2O and brine, dried over anhydrous Na2SO4, evaporated in
a vacuo. The residue was puried by column chromatography
on a silica gel (PE/EtOAc ¼ 15/1–5/1) to provide carbazole 8h.
4.1.3.1 9-((4-(tert-Butyl)phenyl)sulfonyl)-2-(triuoromethyl)-
9H-carbazole (8g). A white solid, 63% yield. 1H NMR (400 MHz,
CDCl3) d 8.65 (s, 1H), 8.37 (d, J ¼ 8.4 Hz, 1H), 8.00 (dd, J ¼ 16.8,
7.9 Hz, 2H), 7.77 (d, J ¼ 8.7 Hz, 2H), 7.63 (d, J ¼ 8.0 Hz, 1H), 7.58
(t, J ¼ 7.9 Hz, 1H), 7.42 (t, J ¼ 7.5 Hz, 1H), 7.37 (d, J ¼ 8.7 Hz,
2H), 1.21 (s, 9H). 13C NMR (100 MHz, CDCl3) d 158.3, 139.3,
137.9, 134.9, 128.8, 126.6, 126.5, 125.2, 124.3, 120.8, 120.5,
115.3, 112.5, 35.4, 31.0 ppm. HRMS calcd for C23H20F3NO2S [M
+ H]+: 432.1245, found: 432.1239.
4.1.3.2 9-((4-Methoxyphenyl)sulfonyl)-2-(triuoromethyl)-9H-
carbazole (8h). A white solid, 69% yield. 1H NMR (400 MHz,
CDCl3) d 8.63 (s, 1H), 8.36 (d, J ¼ 8.5 Hz, 1H), 7.98 (dd, J ¼ 17.1,
8.0 Hz, 2H), 7.76 (d, J ¼ 8.9 Hz, 2H), 7.62 (d, J ¼ 8.0 Hz, 1H), 7.57
(t, J ¼ 7.4 Hz, 1H), 7.41 (t, J ¼ 7.5 Hz, 1H), 6.79 (d, J ¼ 8.9 Hz,
2H), 3.74 (s, 3H). 13C NMR (100 MHz, CDCl3) d 164.2, 139.3,
137.9, 129.4, 129.2, 128.8, 128.7, 125.9, 125.2, 124.3, 123.1,
120.8, 120.5, 115.3, 114.5, 112.6, 112.5, 55.7 ppm. HRMS calcd
for C20H14F3NO3S [M + H]+: 406.0724, found: 406.0721.
Scheme
3 Synthesis of cyclic diphenyliodoniums (5). Reaction
conditions: (a) Pd(PPh3)4, K3PO4, EtOH, reflux, 6 h. (b) (i). NaNO2, 0 ꢂC
THF, 4 M HCl, (ii). KI, rt. (c) mCPBA (85%), TfOH, CH2Cl2, rt, 2 h.
accessible synthesis and the potent anticancer activity may
make these N-arylsulfonyl substituted carbazoles to be prom-
ising drug candidates to kill cancer cells.
4. Experiments
4.1 Chemistry
4.1.1 General information. All solvents were commercially
available and were used without a further purication unless
stated. The chemicals used were either purchased from
commercial sources or prepared according to literature proce-
dures. The 1H and 13C nuclear magnetic resonance (NMR)
spectra were recorded on a Bruker Avance spectrometer 400 at
400 and 100 MHz respectively. Chemical shis are given in ppm
(d) referenced to CDCl3 with 7.26 for 1H and 77.10 for 13C, and to
d6-DMSO with 2.50 for 1H and 39.52 for 13C. In the case of
multiplet, the signals are reported as intervals. Signals are
abbreviated as follows: s, singlet; d, doublet; t, triplet; q, quartet;
m, multiplet. Coupling constants are expressed in hertz. The
progress of the reactions was monitored by thin-layer chroma-
tography on a glass plate coated with silica gel with uorescent
indicator (GF254). Column chromatography was performed on
silica gel (200–300 mesh).
4.1.2 General procedures for the synthesis of cyclic
diphenyleneiodoniums. The procedures11 for the synthesis of
cyclic diphenyliodoniums 5 were depicted in Scheme 3. Cata-
lyzed by Pd(PPh3)4, substituted 2-iodoaniline 1 reacted with
functionalized phenlyboronic acid 2 yielded areneamine 3 at
a good yield. 3 was readily transformed into iodoarene 4 via
a Sandermeyer reaction, and then cyclic diphenyliodoniums 5
could be obtained aer 4 was treated with mCPBA and triic
acid (TfOH) for 2 h at room temperature.
4.1.3.3 9-((4-Fluorophenyl)sulfonyl)-2-(triuoromethyl)-9H-
carbazole (8i). A white solid, 46% yield. 1H NMR (400 MHz,
CDCl3) d 8.61 (s, 1H), 8.34 (d, J ¼ 8.3 Hz, 1H), 7.99 (dd, J ¼ 17.7,
7.9 Hz, 2H), 7.89–7.78 (m, 2H), 7.65 (d, J ¼ 7.9 Hz, 1H), 7.59 (t, J
¼ 7.9 Hz, 1H), 7.44 (t, J ¼ 7.3 Hz, 1H), 7.03 (t, J ¼ 7.8 Hz, 2H). 13
C
NMR (100 MHz, CDCl3) d 167.3, 164.8, 139.2, 137.8, 133.6, 133.6,
129.7, 129.5, 129.4, 129.0, 125.8, 125.4, 124.7, 123.1, 120.9,
120.6, 116.9, 116.7, 115.3, 112.6, 112.5 ppm. HRMS calcd for
C
19H11F4NO2S [M ꢀ H]ꢀ: 392.0369, found: 392.0371.
4.1.3.4 9-((4-Chlorophenyl)sulfonyl)-2-(triuoromethyl)-9H-
carbazole (8j). A white solid, 52% yield. 1H NMR (400 MHz,
CDCl3) d 8.60 (s, 1H), 8.33 (d, J ¼ 8.4 Hz, 1H), 7.99 (dd, J ¼ 18.1,
8.0 Hz, 2H), 7.74 (d, J ¼ 8.5 Hz, 2H), 7.65 (d, J ¼ 8.0 Hz, 1H), 7.58
(t, J ¼ 7.8 Hz, 1H), 7.44 (t, J ¼ 7.5 Hz, 1H), 7.32 (d, J ¼ 8.6 Hz,
2H). 13CNMR (100 MHz, CDCl3) d 141.1, 139.2, 137.8, 136.0,
129.7, 129.4, 129.0, 128.0, 125.4, 124.8, 121.3, 121.3, 121.0,
120.7, 115.3, 112.6, 112.6 ppm. HRMS calcd for C19H11ClF3NO2S
[M + H]+: 410.0029, found: 410.0028.
4.1.3 Synthesis and characterization of the carbazole
derivatives.
4.1.3.5 9-((4-Bromophenyl)sulfonyl)-2-(triuoromethyl)-9H-
carbazole (8k). A white solid, 49% yield. 1H NMR (400 MHz,
CDCl3) d 8.59 (s, 1H), 8.33 (d, J ¼ 8.5 Hz, 1H), 7.99 (dd, J ¼ 17.9,
8.0 Hz, 2H), 7.66 (d, J ¼ 8.7 Hz, 3H), 7.58 (t, J ¼ 7.8 Hz, 1H), 7.48
(d, J ¼ 8.7 Hz, 2H), 7.44 (t, J ¼ 7.5 Hz, 1H). 13C NMR (100 MHz,
CDCl3) d 139.2, 137.8, 136.5, 132.7, 129.7, 129.5, 129.0, 128.0,
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125.5, 124.8, 121.3, 121.0, 120.7, 115.4, 112.6 ppm. HRMS calcd was repeated for three independent times. The percentages of
for C19H11BrF3NO2S [M + H]+: 453.9724, found: 453.9718.
4.1.3.6 2-(Triuoromethyl)-9-((4-(triuoromethyl)phenyl)
sulfonyl)-9H-carbazole (8l). A white solid, 57% yield. 1H NMR
(400 MHz, CDCl3) d 8.61 (s, 1H), 8.35 (d, J ¼ 8.5 Hz, 1H), 8.00
(dd, J ¼ 18.5, 7.9 Hz, 2H), 7.93 (d, J ¼ 8.1 Hz, 2H), 7.67 (d, J ¼
8.1 Hz, 1H), 7.64–7.57 (m, 3H), 7.45 (t, J ¼ 7.6 Hz, 1H). 13C NMR
(100 MHz, CDCl3) d 140.9, 139.1, 137.7, 136.0, 135.7, 129.9,
129.5, 129.1, 127.2, 126.6, 126.6, 125.7, 125.5, 125.0, 124.3,
123.0, 121.5, 121.0, 120.8, 115.3, 112.6, 112.5 ppm. HRMS calcd
for C20H11F6NO2S [M ꢀ H]ꢀ: 442.0337, found: 442.0335.
4.1.3.7 4-((2-(Triuoromethyl)-9H-carbazol-9-yl)sulfonyl)benzo-
nitrile (8m). A white solid, 51% yield. 1H NMR (400 MHz, CDCl3)
d 8.58 (s, 1H), 8.32 (d, J ¼ 8.4 Hz, 1H), 8.00 (dd, J ¼ 18.6, 7.9 Hz,
2H), 7.89 (d, J ¼ 8.6 Hz, 2H), 7.67 (d, J ¼ 8.2 Hz, 1H), 7.63 (dd, J ¼
7.6, 5.0 Hz, 2H), 7.62–7.57 (m, 1H), 7.46 (t, J ¼ 7.6 Hz, 1H). 13C
NMR (100 MHz, CDCl3) d 141.2, 138.9, 137.6, 133.2, 130.0, 129.6,
129.2, 127.2, 125.6, 125.2, 121.7, 121.7, 121.1, 120.8, 118.1, 116.8,
115.3, 112.6, 112.6 ppm. HRMS calcd for C20H11F3N2O2S [M ꢀ
H]ꢀ: 399.0415, found: 399.0415.
cell viability and inhibitory rate of cell viability were calculated
using formulas:
Cell viability % ¼
OD value of treated cells ꢀ OD value of blank
ꢃ 100
OD value of control cells ꢀ OD value of blank
Inhibitory rate of cell viability (%) ¼ 100 ꢀ cell viability (%)
4.2.3 Cell cycle analysis. Cells were seeded in 6-well plates
with a density of 2 ꢃ 105 cells per well and incubated with
different concentrations of drugs for 24 h. The cells were har-
vested and xed with 70% alcohol at 4 ꢂC for 2 h and then
incubated with RNase at 37 ꢂC for 30 min followed by addition
of PI. Finally, cell cycle distribution was detected using FACS
Calibur ow cytometer (BD Biosciences).
4.2.4 Apoptosis assay. Apoptosis was detected by an
Annexin-V/PI assay kit (KeyGEN, Nanjing, China). Aer the drug
treatment, cells were harvested and washed. The cells were then
stained with Annexin-V-FITC and propidium iodide (PI) in
binding buffer for 15 min and apoptotic rates were analyzed
using FACS ow cytometer (BD Biosciences, San Diego, CA,
USA). Comparisons between groups were tested by one-way
ANOVA analysis.
4.2.5 Analysis of ROS. Cells were seeded in six-well plates at
2 ꢃ 105 cells per well overnight and treated with compound 8h
at indicated concentrations for 24 h. Then the cells were
collected and stained with CMH2DCF-DA (Sigma-Aldrich) fol-
lowed by ow cytometry analysis using FACS Calibur ow
cytometer (BD Biosciences). In some experiments of ROS anal-
ysis, cells were pre-treated with 3 mM NAC (Sigma-Aldrich, St.
Louis, MO, USA) for 1 h prior to the exposure to compound 8h.
Statistical comparisons were analyzed by one-way ANOVA.
4.2.6 Western blot analysis. Collected cells were homoge-
nized in lysis buffer (5% SDS, 10 mM EDTA, 50 mM NaCl,
10 mM Tris–HCl). Protein concentrations were measured using
pierce BCA protein assay (Thermo Fisher, Rockford, IL, USA).
Proteins were run on a standard SDS-PAGE and transferred to
PVDF membranes. Membranes were blocked in 5% milk,
incubated with primary antibody at a concentration of 1 : 1000,
then incubated with secondary antibody at a concentration of
1 : 10 000 and read using ECL detection system (Bio-Rad,
Richmond, CA). b-Actin was used as a loading control. Anti-g-
H2AX (phospho-Ser139), anti-cyclinB1, anti-phospho-cdc2
(Tyr15), and anti-b-actin were purchased from Cell Signaling
Technology (Danvers, MA, USA). The expression ratio of
cyclinB1 and p-cdc2 (Tyr15) proteins was quantied against b-
actin using ImageJ soware. One-way ANOVA was used for
statistical analysis.
4.1.3.8 9-(Thiophen-2-ylsulfonyl)-2-(triuoromethyl)-9H-carba-
1
zole (8n). A white solid, 72% yield. H NMR (400 MHz, CDCl3)
d 8.61 (s, 1H), 8.35 (d, J ¼ 8.5 Hz, 1H), 8.00 (dd, J ¼ 16.7, 7.8 Hz,
2H), 7.66 (d, J ¼ 8.1 Hz, 1H), 7.63–7.56 (m, 2H), 7.48–7.42 (m,
2H), 6.92 (dd, J ¼ 5.0, 3.9 Hz, 1H). 13C NMR (100 MHz, CDCl3)
d 139.0, 137.6, 136.9, 133.4, 132.9, 129.5, 129.2, 128.8, 127.5,
125.7, 125.4, 124.7, 123.0, 121.1, 120.7, 120.4, 115.5, 112.8 ppm.
HRMS calcd for C17H10F3NO2S2 [M + H]+: 382.0183, found:
382.0181.
4.2 Biological assay
4.2.1 Cell lines and cell culture. Human cell lines Capan-2
(pancreatic cancer) and HCT-116 (colon cancer) were purchased
from American Tissue Culture Collection (Manassas, VA, USA).
Cell lines PANC-1 (pancreatic cancer), SGC-7901 (gastric
cancer), HL-60 (leukemia) and HFL-1 (normal human lung
broblast) were purchased from the cell bank, Chinese
Academy of Sciences (Shanghai, China). The medium for cell
culture were specied by the instructions of cell repository
banks. All medium were obtained from Invitrogen Life Tech-
nologies (Carlsbad, CA, USA) and supplemented with 10% fetal
bovine serum (GIBCO, Carlsbad, CA, USA). All cell lines were
cultured at 37 ꢂC in a humidied incubator with 5% CO2
atmosphere.
4.2.2 MTS assay. Cell viability was determined by MTS
assay (Promega, Madison, WI, USA).36 Cells were seeded in a 96-
well plate at a density of 3000 cells per well. We simultaneously
prepared blank wells containing media only. The stock solu-
tions of test compounds were dissolved in appropriate volume
so that the nal DMSO concentration during treatment in cells
was not more than 0.1%. Treated cells were cultured with
indicated compounds while control cells were treated with 0.1%
(v/v) DMSO. Aer 72 h of treatment, 20 mL MTS reagent was
added to each well and incubated for an additional 4 hours at
4.2.7 Animal study. The exponentially growing PANC-1 and
Capan-2 cells were collected and injected subcutaneously into
the anks of 5 week-old female immune-decient BALB/c nude
mice (Vital River, Beijing, China) at 2 ꢃ 106 cells or 3 ꢃ 106 cells
ꢂ
37 C. The optical density (OD) was determined by microplate
reader (Thermo, Helsinki, Finland) at 490 nm. The experiment
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per injection site respectively. Aer the formation of palpable
tumors, the nude mice were randomly assigned into control and
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values were calculated using the two-tailed Student t-test. The 14 Z. Liu, D. Zhu, B. Luo, N. Zhang, Q. Liu, Y. Hu, R. Pi,
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of Sun Yat-sen University Cancer Center. The code for IACUC
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20 H. Xie, M. Ding, M. Liu, T. Hu and F. Zhang, Org. Lett., 2017,
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Conflicts of interest
There are no conicts to declare.
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22 In Anticancer Drug Development Guide: Preclinical Screening,
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Acknowledgements
This work was supported in part by National Natural Science
Foundation of China (81672952, 81430060), and Guangdong
Science and Technology Program (2017A020215198).
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